At-line determination of the enantiomeric excess in multi-component chiral samples using Raman Optical Activity (ROA)

Background: Enantiopure drugs offer better specificity, higher therapeutic index, and optimized pharmacokinetics compared to racemic drugs. However, pharmaceutical companies continue manufacturing racemic medicines, among others, due to the lack of tools for in situ determination of the optical purity and the absolute configura-tion, which is necessary for approval of drug substance by the European Medicines Agency (EMA) or the US Food and Drug Administration (FDA). Currently used methods for the absolute configuration and enantiomeric excess determination have significant limitations (require single crystal, UV-active chromophores, chiral reagents, etc). Results: Addressing this need of pharmacological industry for the effective analysis of chiral drugs in situ in solu-tion, we have designed and constructed the at-line setup based on Raman Optical Activity (ROA) and equipped with the 3D-printed flow reactor dedicated for fast in situ and in-flow analysis of chiral compounds. We have validated our approach by the determination of the enantiomeric excess (EE) of pure and multi-component chiral samples showing that the developed solution combines the excellent stereosensitivity, possibility to measure chi-ral samples in-flow, and a rather unique capability of direct discrimination of components of chiral mixtures. The quantitative analysis based on Partial Least Squares (PLS) regression has demonstrated that the accuracy and pre-cision of the method is very high with the average prediction error from cross-validation (RMSECV) not exceeding 1.35% (for R2 at least 99.95) for ROA spectra recorded in 20 minutes. Significance: According to our best knowledge, our work is a first example of successful in-flow and in situ ROA evaluation of chiral purity in chiral mixtures. Our direct approach does not require sample preparation, preliminary separation of enantiomers or derivatization. In the long-term, our solution paves the way toward operando (in-line) ROA, enabling real-time monitoring of chirality in reaction mixtures.